Defects in p53 signaling eliminate apoptotic responses to radiation therapy in many human cancers. In head and neck squamous cell carcinoma (HNSCC), the fifth most common cancer worldwide, TP53 mutations cause locoregional recurrence of radioresistant tumors, an invariably fatal form of the disease. Thus there is an urgent need for agents that will bypass mutant TP53 to restore radiosensitivity in HNSCC. This proposal focuses on an emerging apoptotic pathway, designated 'Chk1-suppressed' (CS) apoptosis, whose activation by Chk1 inhibitors restores radiosensitivity in p53-deficient zebrafish, mouse, and human cancer cells (Sidi et al., Cell 2008). We propose that Chk1 inhibitors and associated CS pathway define a promising therapeutic opportunity for TP53 mutant HNSCC. Our work has elucidated the core backbone of the CS pathway, which comprises a novel ATM/ATR-caspase-2 axis that bypasses p53 and attendant mitochondrial and death-receptor signaling cascades. Recently, we identified the PIDDosome (PIDD-RAIDD-caspase-2 complex), but not the intrinsic apoptosome or extrinsic DISC, as the caspase-activation platform at work in the CS pathway. These results strengthen the notion that the CS axis defines a third apoptotic pathway in vertebrate cells and were published in the September 14th issue of Molecular Cell (Ando et al. Mol Cell 2012). While we hypothesize that the CS pathway will be therapeutically effective in TP53 mutant HNSCC, the extreme heterogeneity of this disease makes it essential that we develop biologic tools that predict or assess PIDDosome activity in tumors. However, our molecular understanding of PIDDosome biology is very limited. To both deepen our understanding of PIDDosome signaling and identify predictive and pharmacodynamic biomarkers of CS pathway therapy, we propose three specific aims. The first and second aims are designed to identify novel PIDDosome regulators and substrates, respectively, by elucidating the roles of four PIDD-interacting molecules we recently identified. The third aim integrates cutting-edge cancer genomics with in vivo functional genetics in zebrafish to identify genetic predictors of HNSCC response to CS pathway therapy. Candidate predictive genotypes will be functionally characterized using the functional CS pathway markers identified in Aim 1, Aim 2, or our previous studies, and validated in ex vivo cultures of primary HNSCC samples from the OR. In summary, we aim to make a significant impact in the newly emerging area of PIDDosome-mediated apoptotic signaling, thereby translating CS apoptosis into an effective HNSCC therapy.